INTERACTION OF DNA WITH CATIONIC VESICLES FROM AMPHIPHILIC METAL COMPLEXES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。阳离子脂质体能促进DNA跨细胞膜转移，被认为是潜在的非病毒DNA载体，可用于基因治疗。高分辨同步辐射X射线衍射仪研究表明，脂质体基因的转导效率与DNA与阳离子囊泡的相互作用机制有关。为了设计一种既能通过静电作用又能通过氢键与DNA相互作用的阳离子脂质体，我们合成了一种新型的两亲性金属络合物，这种络合物能够在水介质中形成囊泡，具有高的阳离子电荷密度和明确的氢键基序，适合与DNA的磷酸基团结合。新形成的囊泡已被证明有效地将绿色荧光蛋白(GFP)pEGFP-N1(4.9kb)导入哺乳动物HEK 293-T细胞系。动态光散射(DLS)研究表明，当它们与线性DNA络合时，脂质体的直径从400 nm增加到800 nm。透射电子显微镜研究表明，阳离子脂质体与DNA相互作用形成洋葱状结构，其中DNA单层夹在阳离子脂双层之间。为了证实这一观察结果，我们建议使用小角X射线散射(SAXS)，我们希望看到插入的DNA层产生布拉格反射。对合成的阳离子脂质体与DNA相互作用的研究有助于为脂质体辅助细胞转导的机制提供新的理论基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cationic liposomes had been reported to facilitate DNA transfer across cell membranes and are considered to be potential non-viral DNA carriers for gene therapy. High-resolution synchrotron x-ray diffraction (XRD) studies show that the efficiency of the liposome gene transfection is associated with the interaction mechanism between DNA and the cationic vesicles. In an effort to rationally design a cationic liposome that could interact with DNA not only by electrostatic forces but by hydrogen bonding, we synthesized novel amphiphilic metal complexes which were able to form vesicles in aqueous media with high cationic charge densities and well-defined hydrogen bonding motifs suitable for binding to the phosphate backbone groups of DNA. The newly formed vesicles have been proved effective for the transfection of the Green Fluorescent Protein (GFP) pEGFP-N1 (4.9 kb) into mammalian HEK 293-T cell lines. Dynamic light scattering (DLS) studies show a liposome diameter increase from 400 nm to 800 nm when they are complexed with linear DNA. Studies done by transmission electron microscopy (TEM) suggest that the cationic liposome interacts with DNA forming an onion-like structure where DNA monolayers are sandwiched between cationic lipid bilayers. In order to confirm this observation we propose to use small angle x-ray scattering (SAXS) where we expect to see the Bragg reflections result from the intercalated DNA layer. The study of the interactions between DNA and the synthetic cationic liposome could help to develop a new theory on the mechanism of liposome assisted cell transfection.